FGFR3 is one of four members of the FGFR family of transmembrane tyrosine kinase receptors that are involved in the intracellular signaling pathways. Activation of FGFR through interaction with fibroblast growth factors (FGFs) has been shown to have a critical role in both embryogenesis and adults with a pleiotropic range of sequelae, including cell proliferation and survival, migration, differentiation and growth arrest [1].
FGFRs consist of an extracellular ligand binding region, with two or three immunoglobulin-like domains (IgD1-3), a single-pass transmembrane region, and a cytoplasmic, split tyrosine kinase domain.
In humans, signaling is mediated by 1 of 22 FGF ligands, and ligand receptor specificity is controlled by differential cellular expression of the receptors, secretion of cell-surface proteins that modulate the interaction and alternative splicing of the receptors [1]. FGFR1, 2 and 3 each have two major alternatively spliced isoforms, designated IIIb and IIIc. These isoforms differ by about 50 amino acids in the second half of IgD3, and have distinct tissue distribution and ligand specificity. FGF ligands cause dimerization of FGFRs, which leads to activation and phosphorylation of the intracellular tyrosine kinase domain. This leads to activation of the several key pathways implicated in oncogenic signaling, including the mitogen-activated protein kinase (MAPK) and PI3K-AKT pathways.
Aberrantly activated FGFRs have been implicated in specific human malignancies [2]. In particular, the t(4; 14) (p16.3; q32) chromosomal translocation occurs in about 15-20% of multiple myeloma patients, leading to overexpression of FGFR3 and correlates with shorter overall survival [2]. FGFR3 is implicated also in conferring chemoresistance to myeloma cell lines in culture [3], consistent with the poor clinical response of t(4; 14) patients to conventional chemotherapy [4]. Overexpression of mutationally activated FGFR3 is sufficient to induce oncogenic transformation in hematopoietic cells and fibroblasts [5-8], transgenic mouse models [9], and murine bone marrow transplantation models [9, 10].
Accordingly, FGFR3 has been proposed as a potential therapeutic target in multiple myeloma. Indeed, several small-molecule inhibitors targeting FGFRs, although not selective for FGFR3 and having cross-inhibitory activity toward certain other kinases, have demonstrated cytotoxicity against FGFR3-positive myeloma cells in culture and in mouse models [11-15].
FGFR3 overexpression has been documented also in a high fraction of bladder cancers [16, 17]. Furthermore, somatic activating mutations in FGFR3 have been identified in 60-70% of papillary and 16-20% of muscle-invasive bladder carcinomas [17, 18]. In cell culture experiments, RNA interference [19] or an FGFR3 single-chain Fv antibody fragment inhibited bladder cancer cell proliferation [20]. A recent study demonstrated that an FGFR3 antibody-toxin conjugate attenuates xenograft growth of a bladder cancer cell line through FGFR3-mediated toxin delivery into tumors [21]. Publications relating to FGFR3 and anti-FGFR3 antibodies include US 2005/0147612; WO 2010/111367; Rauchenberger et al, J Biol Chem 278 (40):38194-38205 (2003)[22]; WO 2006/048877; Martinez-Torrecuadrada et al, (2008) Mol Cancer Ther 7(4): 862-873; WO 2007/144893; Trudel et al. (2006) 107(10): 4039-4046; Martinez-Torrecuadrada et al (2005) Clin Cancer Res 11 (17): 6280-6290; Gomez-Roman et al (2005) Clin Cancer Res 11:459-465; and WO 2010/002862.
These antibodies have one or more of the following disadvantages: they have the capability to recognize only one of the isoforms, or display a significant difference between affinities for the different isoforms.
Thus, there remains a need for molecules that can bind both splice variants FGFR3b and FGFR3c, with high affinity and specificity, and that are at the same time capable of being well internalized into a cell.
This need is addressed by the present invention.